Noval madcam antibodies

ABSTRACT

The invention provides new, improved anti-MAdCAM antibodies. Uses of these antibodies in medicine are also included, in particular for the treatment of inflammatory conditions such as inflammatory bowel disease.

This application is a continuation of U.S. patent application Ser. No. 11/484,456, filed Jul. 10, 2006, which claims priority, under 35 U.S.C. §119(e), from U.S. Provisional Application No. 60/697,453, filed Jul. 8, 2005.

FIELD OF THE INVENTION

The invention relates to an improved human antibody specific for Mucosal addressin cell adhesion molecule (MAdCAM).

BACKGROUND OF THE INVENTION,

Mucosal addressin cell adhesion molecule (MAdCAM) is a member of the immunoglobulin superfamily of cell adhesion receptors. It is one of the adhesion molecules involved in the recruitment of lymphocytes to tissues when required, by means of interacting with an integrin molecule on the surface of the lymphocytes.

It has been shown that antibodies that inhibit binding of MAdCAM to its integrin binding partner, α₄β7 for example anti-MAdCAM antibodies (e.g. MECA-367; U.S. Pat. No. 5,403,919, U.S. Pat. No. 5,538,724) or anti-α₄β₇ antibodies (e.g. Act-1; U.S. Pat. No. 6,551,593 or humanised Act-1, also called MLN02, described in WO 01/78779), can inhibit leukocyte extravasation into inflamed intestine, and can therefore be beneficial in the treatment of inflammatory bowel disease.

Anti-MAdCAM antibodies such as MECA-367, however, are not therapeutically useful in human patients; MECA-367 binds mouse MAdCAM, and does not show much affinity for the human MAdCAM molecule. In addition, being a rat antibody, it will lead to an immune response in human patients and therefore not be suitable for therapeutic use. Mouse monoclonal antibodies, directed against human MAdCAM have been described (WO 96/24673), but these are also likely to be immunogenic in humans. Recently, therapeutically useful, fully human anti-human MAdCAM antibodies with exquisite specificity and affinity to human and primate MAdCAM have been developed and disclosed in WO2005/067620.

It is known that even fully human antibodies can still lead to an immune response in some patients, which is likely to be due to sequences in the variable region of the antibody. The antibodies of the present invention are modifications of the human anti-MAdCAM antibodies disclosed in WO2005/067620, which are likely to be even less prone to any immunogenicity problems in human patients.

SUMMARY OF THE INVENTION

One aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the heavy chain of 6.22.2-mod_V (SEQ ID NO: 2).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the light chain of 6.34.2-mod_SSQ, 6.34.2-mod_QT, or 6.34.2-mod_SSQ,QT (SEQ ID NOs: 8, 10, 12).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the light chain of 6.67.1-mod_Y, 6.67.1-mod_TΔP, or 6.67.1-mod_Y,TΔP (SEQ ID NOs: 16, 18, 20).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the heavy chain of 6.77.1-mod_ΔS (SEQ ID NO: 22).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the light chain of 7.16.6_V, 7.16.6_S, or 7.16.6_VS (SEQ ID NOs: 30, 32, 34).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the heavy chain of 7.16.6_L (SEC) ID NO: 28).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the light chain of 7.16.6_V, 7.16.6_S, or 7.16.6_VS (SEQ ID NOs: 30, 32, 34) and the variable region of the heavy chain of 7.16.6_L (SEQ ID NO: 28).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the heavy chain of 9.8.2_ΔRGAYH,D (SEQ ID NO: 36).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, comprising the variable region of the light chain of 9.8.2-mod (SEQ ID NO: 38).

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, wherein the antibody is selected from the group consisting of:

(a) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3;

(b) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 5 and SEQ ID NOs: 7, 9 or 11;

(c) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 13 and SEQ ID NOs: 15, 17, or 19;

(d) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 23;

(e) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NOs: 25 or 27 and SEQ ID NOs: 29, 31, or 33; or

(f) an antibody comprising the variable regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 35 and SEQ ID NO: 37.

Another aspect of the invention is a monoclonal antibody that specifically binds MAdCAM, wherein the antibody is selected from the group consisting of:

(a) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4, without the signal sequences;

(b) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NOs: 8, 10, or 12, without the signal sequences;

(c) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NO: 14 and SEQ ID NOs: 16, 18, or 20, without the signal sequences;

(d) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24, without the signal sequences;

(e) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NOs: 26 or 28 and SEQ ID NOs: 30, 32 or 34, without the signal sequences; or

(f) an antibody comprising the variable regions of the amino acid sequences set forth in SEQ ID NO: 36 and SEQ ID NO: 38, without the signal sequences.

Another aspect of the invention is a monoclonal antibody or antigen-binding portion thereof, wherein said antibody comprises:

(a) a heavy chain of amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 4, without the signal sequences.

(b) a heavy chain of amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NOs: 8, 10, or 12, without the signal sequences.

(c) a heavy chain of amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NOs: 16, 18, or 20, without the signal sequences.

(d) a heavy chain of amino acid sequence of SEQ ID NO: 22 and the light chain amino acid sequence of SEQ ID NO: 24, without the signal sequences.

(e) a heavy chain of amino acid sequence of SEQ ID NOs: 26 or 28 and the light chain amino acid sequence of SEQ ID NOs: 30, 32 or 34, without the signal sequences.

(f) a heavy chain of amino acid sequence of SEQ ID NO: 36 and the light chain amino acid sequence of SEQ ID NO: 38, without the signal sequences.

Another aspect of the invention is a human monoclonal antibody of the invention, wherein the heavy chain C-terminal lysine is cleaved.

Another aspect of the invention is a human monoclonal antibody of the invention, which is an IgG₂ isotype. A further aspect of the invention is a human monoclonal antibody of the invention, which is an IgG₄ isotype. A further aspect of the invention is a human monoclonal antibody of the invention which comprises a is light chain. A further aspect of the invention is a human monoclonal antibody of the invention which comprises a light chain. A preferred aspect of the invention is a human monoclonal antibody of the invention which is an IgG₂ isotype with a κ light chain.

Another aspect of the invention is a vector comprising any of the sequences of the variable regions of SEQ ID NOs: 1, 7, 9, 11, 15, 17, 19, 21, 27, 29, 31, 33, 35, or 37 or any of the sequences encoding the variable regions of SEQ ID NOs: 2, 8, 10, 12, 16, 18, 20, 22, 28, 30, 32, 34, 36 or 38. A further aspect of the invention is a host cell comprising any of the sequences of the variable regions of SEQ ID NOs: 1, 7, 9, 11, 15, 17, 19, 21, 27, 29, 31, 33, 35 or 37, or any of the sequences encoding the variable regions of SEQ ID NOs: 2, 8, 10, 12, 16, 18, 20, 22, 28, 30, 32, 34, 36 or 38. The host cell can be a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or Pichia pistoris, a plant cell, an insect cell, or a mammalian cell. More preferably, the host cell is a mammalian cell. Most preferably, the host cell is a CHO cell or NS0 cell.

Another aspect of the invention is a method of producing the antibody of the invention, comprising culturing a host cell comprising any of the sequences of the variable regions of SEQ ID NOs: 1, 7, 9, 11, 15, 17, 19, 21, 27, 29, 31, 33, 35 or 37, or any of the sequences encoding the variable regions of SEQ ID NOs: 2, 8, 10, 12, 16, 18, 20, 22, 28, 30, 32, 34, 36 or 38, under conditions in which the antibody is expressed, and recovering the antibody from the host cell or its culture supernatant.

Another aspect of the invention is a transgenic animal or a transgenic plant, comprising any of the sequences of the variable regions of SEQ ID NOs: 1, 7, 9, 11, 15, 17, 19, 21, 27, 29, 31, 33, 35 or 37, or any of the sequences encoding the variable regions of SEQ ID NOs: 2, 8, 10, 12, 16, 18, 20, 22, 28, 30, 32, 34, 36 or 38.

One aspect of the invention is the use of the antibodies of the invention or an antigen-binding portion thereof for use as a medicament. Another aspect of the invention is the use of the antibodies of the invention or an antigen-binding portion thereof for the manufacture of a medicament for the treatment of conditions involving MAdCAM-mediated adhesion of leukocytes. Another aspect of the invention is the use of the antibodies of the invention or an antigen-binding portion thereof for the treatment of inflammatory conditions, such as but not limited to inflammatory diseases of the gastrointestinal tract including inflammatory bowel disease such as Crohn's disease, ulcerative colitis; diverticula disease, gastritis, liver disease, primary sclerosis, sclerosing cholangitis. Inflammatory diseases also include but are not limited to abdominal disease (including peritonitis, appendicitis, biliary tract disease), acute transverse myelitis, allergic dermatitis (including allergic skin, allergic eczema, skin atopy, atopic eczema, atopic dermatitis, cutaneous inflammation, inflammatory eczema, inflammatory dermatitis, flea skin, miliary dermatitis, miliary eczema, house dust mite skin), ankylosing spondylitis (Reiters syndrome), asthma, airway inflammation, atherosclerosis, arteriosclerosis, biliary atresia, bladder inflammation, breast cancer, cardiovascular inflammation (including vasculitis, rheumatoid nail-fold infarcts, leg ulcers, polymyositis, chronic vascular inflammation, pericarditis, chronic obstructive pulmonary disease), pancreatitis, including chronic pancreatitis, perineural inflammation, coeliac disease, colitis (including amoebic colitis, infective colitis, bacterial colitis, Crohn's colitis, ischemic colitis, ulcerative colitis, idiopathic proctocolitis, inflammatory bowel disease, pseudomembranous colitis), chloroma, collagen vascular disorders (rheumatoid arthritis, SLE, progressive systemic sclerosis, mixed connective tissue disease, diabetes mellitus), Crohn's disease (regional enteritis, granulomatous ileitis, ileocolitis, digestive system inflammation), demyelinating disease (including myelitis, multiple sclerosis, disseminated sclerosis, acute disseminated encephalomyelitis, perivenous demyelination, vitamin B12 deficiency, Guillain-Barre syndrome, MS-associated retrovirus), dermatomyositis, diverticulitis, emphysema, exudative diarrhea, fever in immunocompromised patients, gastritis, granulomatous hepatitis, granulomatous inflammation, cholecystitis, insulin-dependent diabetes mellitus, liver inflammatory diseases (liver fibrosis including but not limited to Hepatitis C-induced liver damage, alcoholic liver disease, non-alcoholic steatohepatitis (NASH); primary biliary cirrhosis, hepatitis, sclerosing cholangitis), lung inflammation (idiopathic pulmonary fibrosis, eosinophilic granuloma of the lung, pulmonary histiocytosis X, peribronchiolar inflammation, acute bronchitis), lymphogranuloma venereum, malignant melanoma, mouth/tooth disease (including gingivitis, periodontal disease), metastatic cancers, mucositis, musculoskeletal system inflammation (myositis), nonalcoholic steatohepatitis (nonalcoholic fatty liver disease), ocular & orbital inflammation (including uveitis, optic neuritis, peripheral rheumatoid ulceration, peripheral corneal inflammation,), osteoarthritis, osteomyelitis, pharyngeal inflammation, polyarthritis, proctitis, psoriasis, radiation injury, sarcoidosis, sickle cell necropathy, superficial thrombophlebitis, systemic inflammatory response syndrome, thyroiditis, systemic lupus erythematosus, tropical sprue, graft versus host disease, acute burn injury, Behçet's syndrome, Sjögren's syndrome, uterine disorders such as endometriosis, dysmenorrhea or pelvic inflammatory disease. Preferably, the antibodies of the invention are used for the treatment of inflammatory bowel disease such as Crohn's disease and ulcerative colitis, graft versus host disease, liver fibrosis, uveitis. Even more preferably, the antibodies of the invention are used for the treatment is of inflammatory bowel disease.

Another aspect of the invention is a method of treatment of a condition mentioned above, comprising administering to a patient in need of such treatment a therapeutically effective amount of an antibody of the invention.

Another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention or an antigen-binding portion thereof and a pharmaceutically acceptable carrier or excipient.

DETAILED DESCRIPTION OF THE INVENTION

A nucleic acid molecule encoding the entire heavy chain of an anti-MAdCAM antibody of the invention may be constructed by fusing a nucleic acid molecule encoding the entire variable domain of a heavy chain or an antigen-binding domain thereof with a constant domain of a heavy chain. Similarly, a nucleic acid molecule encoding the light chain of an anti-MAdCAM antibody may be constructed by fusing a nucleic acid molecule encoding the variable domain of a light chain or an antigen-binding domain thereof with a constant domain of a light chain. Nucleic acid molecules encoding the VH and VL regions may be converted to full-length antibody genes by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the heavy chain constant region (CH) segment(s) within the vector and the VL segment is operatively linked to the light chain constant region (CL) segment within the vector. Alternatively, the nucleic acid molecules encoding the VH or VL chains are converted into full-length antibody genes by linking, e.g., ligating, the nucleic acid molecule encoding a VH chain to a nucleic acid molecule encoding a CH chain using standard molecular biological techniques. The same may be achieved using nucleic acid molecules encoding VL and CL chains. The sequences of human heavy and light chain constant region genes are known in the art. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242 (1991). Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-MAdCAM antibody isolated.

A nucleic acid molecule encoding either the heavy chain of an anti-MAdCAM antibody of the invention or an antigen-binding portion thereof, or the light chain of an anti-MAdCAM antibody of the invention or an antigen-binding portion thereof may be prepared, for example, by synthesizing appropriate nucleic acids using the sequence information provided herein, and splicing them together with the desired constant regions of immunoglobulin genes. The skilled person will be well aware of the appropriate methods.

The nucleic acid molecules may be used to recombinantly express large quantities of anti-MAdCAM antibodies, as described below. The nucleic acid molecules may also be used to produce chimeric antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization, also as described below.

The invention provides vectors comprising the nucleic acid molecules of the invention that encode the heavy chain or the antigen-binding portion thereof. The invention also provides vectors comprising the nucleic acid molecules of the invention that encode the light chain or antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

To express the antibodies, or antibody portions used in the invention, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615, each of which is hereby incorporated by reference. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants are known in the art. See, e.g, U.S. Pat. No. 6,517,529. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors used in the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr⁺ host cells with methotrexate selection/amplification) and the neo gene (for G418 selection), and the glutamate synthetase gene.

Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of an anti-MAdCAM antibody, and vectors comprising these nucleic acid molecules, can be used for transformation of a suitable mammalian plant, bacterial or yeast host cell. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods of transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NS0, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof, the light chain and/or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coil and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Further, expression of antibodies used in the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, 0 338 841 and 0 323 997.

It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies.

Transgenic non-human animals and transgenic plants comprising one or more nucleic acid molecules described above may be used to produce antibodies used in the invention. Antibodies can be produced in and recovered from tissue or bodily fluids, such as milk, blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957. As described above, non-human transgenic animals that comprise human immunoglobulin loci can be immunized with MAdCAM or a portion thereof. Methods for making antibodies in plants are described, e.g., in U.S. Pat. Nos. 6,046,037 and 5,959,177, incorporated herein by reference.

Non-human transgenic animals and transgenic plants are produced by introducing one or more nucleic acid molecules used in the invention into the animal or plant by standard transgenic techniques. See Hogan, supra. The transgenic cells used for making the transgenic animal can be embryonic stem cells, somatic cells or fertilized egg cells. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999). The transgenic non-human organisms may have a targeted disruption and replacement that encodes a heavy chain and/or a light chain of interest. The transgenic animals or plants may comprise and express nucleic acid molecules encoding heavy and light chains that combine to bind specifically to MAdCAM, preferably human MAdCAM. The transgenic animals or plants may comprise nucleic acid molecules encoding a modified antibody such as a single-chain antibody, a chimeric antibody or a humanized antibody. The anti-MAdCAM antibodies may be made in any transgenic animal. The non-human animals include mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other bodily fluids.

Treatment may involve administration of one or more inhibitory anti-MAdCAM monoclonal antibodies of the invention, or antigen-binding fragments thereof, alone or with a pharmaceutically acceptable carrier. Inhibitory anti-MAdCAM antibodies and compositions comprising them, can be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. Such additional agents may be included in the same composition or administered separately. Additional therapeutic agents include anti-inflammatory or immunomodulatory agents. These agents include, but are not limited to, the topical and oral corticosteroids such as prednisolone, methylprednisolone, NCX-1015 or budesonide; the aminosalicylates such as mesalazine, olsalazine, balsalazide or NCX-456; the class of immunomodulators such as azathioprine, 6-mercaptopurine, methotrexate, cyclosporin, FK506, IL-10 (Ilodecakin), IL-11 (Oprelevkin), IL-12, MIF/CD74 antagonists, CD40 antagonists, such as TNX-100/5-D12, OX40L antagonists, GM-CSF, pimecrolimus or rapamycin; the class of anti-TNFα agents such as infliximab, adalimumab, CDP-870, onercept, etanercept; the class of anti-inflammatory agents, such as PDE-4 inhibitors (roflumilast, etc), TACE inhibitors (DPC-333, RDP-58, etc) and ICE inhibitors (VX-740, etc) as well as IL-2 receptor antagonists, such as daclizumab, the class of selective adhesion molecule antagonists, such as natalizumab, MLN-02, or alicaforsen, classes of analgesic agents such as, but not limited to, COX-2 inhibitors, such as rofecoxib, valdecoxib, celecoxib, P/Q-type voltage sensitive channel (α2δ) modulators, such as gabapentin and pregabalin, NK-1 receptor antagonists, cannabinoid receptor modulators, and delta opioid receptor agonists, as well as anti-neoplastic, anti-tumor, anti-angiogenic chemotherapeutic agents, or anti-fibrotic agent including but not limited to protease inhibitors, preferably caspase inhibitors, more preferably the caspase inhibitor is a compound of formula I.

The Compound of Formula I

wherein A, B, R1 and R2 are as defined below.

wherein

A is a natural or unnatural amino acid of Formula IIa-i:

B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH₂)_(n)-cycloalkyl, (CH₂)_(n)-phenyl, (CH₂)_(n)-(substituted phenyl), (CH₂)_(n)-(1 or 2-naphthyl), (CH₂)_(n)-(substituted 1 or 2-naphthyl), (CH₂)_(n)-(heteroaryl), (CH₂)_(n)-(substituted heteroaryl), halomethyl, CO₂R¹², CONR¹³R¹⁴, CH₂ZR¹⁵, CH₂OCO(aryl), CH₂OCO(heteroaryl), or CH₂OPO(R¹⁶)R¹⁷, where Z is an oxygen or a sulfur atom, or B is a group of the Formula IIIa-c:

R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, substituted (1 or 2 naphthyl)alkyl, heteroaryl, substituted heteroaryl, (heteroaryl)alkyl, substituted (heteroaryl)alkyl, R^(1a)(R^(1b))N, or R^(1c)O; and

R² is hydrogen, lower alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, or substituted (1 or 2 naphthyl)alkyl;

And wherein:

R^(1a) and R^(1b) are independently hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, substituted (1 or 2 naphthyl)alkyl, heteroaryl, substituted heteroaryl, (heteroaryl)alkyl, or substituted (heteroaryl)alkyl, with the proviso that R^(1a) and R^(1b) cannot both be hydrogen;

R^(1c) is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, substituted (1 or 2 naphthyl)alkyl, heteroaryl, substituted heteroaryl, (heteroaryl)alkyl, or substituted (heteroaryl)alkyl;

R³ is C₁₋₆ alkyl, cycloalkyl, phenyl, substituted phenyl, (CH₂)_(n)NH₂, (CH₂)NHCOR⁹, (CH₂)_(n)N(C═NH)NH₂, (CH₂)_(m)CO₂R², (CH₂)_(m)OR¹⁰, (CH₂)_(m)SR¹¹, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl) or (CH₂)_(n)(heteroaryl), wherein heteroaryl includes pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, pyrazinyl, pyrimidyl, triazinyl, tetrazolyl, and indolyl;

R^(3a) is hydrogen or methyl, or R³ and R^(3a) taken together are —(CH₂)_(d)— where d is an integer from 2 to 6;

R⁴ is phenyl, substituted phenyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), cycloalkyl, or benzofused cycloalkyl;

R⁵ is hydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R⁶ is hydrogen, fluorine, oxo, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), OR¹⁰, SR¹¹, or NHCOR⁹;

R⁷ is hydrogen, oxo (i.e. ═O), lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R⁸ is lower alkyl, cycloalkyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), or COR⁹;

R⁹ is hydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), OR¹², or NR¹³R¹⁴;

R¹⁰ is hydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R¹¹ is lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R¹² is lower alkyl, cycloalkyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R¹³ is hydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl);

R¹⁴ is hydrogen or lower alkyl; or

R¹³ and R¹⁴ taken together form a five to seven membered carbocyclic or heterocyclic ring, such as morpholine, or N-substituted piperazine;

R¹⁵ is phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), or (CH₂)_(n)(heteroaryl);

R¹⁶ or R¹⁷ are independently lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, phenylalkyl, substituted phenylalkyl, or (cycloalkyl)alkyl;

R¹⁸ and R¹⁹ are independently hydrogen, alkyl, phenyl, substituted phenyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or R¹⁸ and R¹⁹ taken together are —(CH═CH)₂—;

R²⁰ is hydrogen, alkyl, phenyl, substituted phenyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl);

R²¹, R²² and R²³ are independently hydrogen, or alkyl;

X is CH₂, (CH₂)₂, (CH₂)₃, or S;

Y¹ is O or NR²³;

Y² is CH₂, O, or NR²³;

a is 0 or 1 and b is 1 or 2, provided that when a is 1 then b is 1;

c is 1 or 2, provided that when c is 1 then a is 0 and b is 1;

m is 1 or 2; and

n is 1, 2, 3, or 4;

or a pharmaceutically acceptable salt thereof.

As used herein, the term “alkyl” means a straight or branched C₁ to C₁₀ carbon chain, such as methyl, ethyl, tert-butyl, iso-propyl, n-octyl, and the like. The term “lower alkyl” means a straight chain or branched C₁ to C₆ carbon chain, such as methyl, ethyl, iso-propyl, and the like.

Depending on the choice of solvent and other conditions known to the skilled person, these compounds may also take the ketal or acetal form, and the use of these forms in the combination of the invention is included in the invention.

These compounds can be prepared as described in WO 00/01666 or in U.S. Pat. No. 6,544,951, hereby incorporated by reference in their entirety. Preferred subgroups are those listed in U.S. Pat. No. 6,544,951.

Preferred are compounds selected from:

(3S)-3-[N—(N′-(2-Fluoro-4-Iodophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Chlorophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Bromophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Fluorophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Trifluoromethylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(1-Anthryl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Trifluoromethylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2,6-Difluorophenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(1-Naphthyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(4-Methoxyphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid;

(3S)-3-[N—(N′-(2-Trifluoromethylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic acid;

(3S)-3-[N—(N′-(2-tert-Butylmethylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic acid;

(3S)-3-[N—(N′-(2-Benzylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic acid;

(3S)-3-[N—(N′-(2-Phenylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic acid.

Most preferably, the compound is (3S)-3-[N—(N′-(2-Tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic acid.

As used herein, “pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, Polyethylene glycol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are surfactants, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

The compositions used in this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, lyophilized cake, dry powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intradermal). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular, intradermal or subcutaneous injection. If desired, the antibody may be administered by using a pump, enema, suppository, or indwelling reservoir or such like.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, lyophilized cake, dry powder, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the anti-MAdCAM antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile solution thereof. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. The desired characteristics of a solution can be maintained, for example, by the use of surfactants and the required particle size in the case of dispersion by the use of surfactants, phospholipids and polymers. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts, polymeric materials, oils and gelatin.

The antibodies of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, intradermal or intravenous infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

In certain embodiments, the antibody compositions may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978)).

In certain embodiments, an anti-MAdCAM antibody of the invention can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the anti-MAdCAM antibodies can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

The compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antigen-binding portion of the invention. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a pre-determined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the anti-MAdCAM antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In some embodiments, a formulation contains 5 mg/mL of antibody in a buffer of 20 mM sodium acetate, pH 5.5, 140 mM NaCl, and 0.2 mg/mL polysorbate 80. In other embodiments, a formulation contains 10 mg/ml of antibody in 2.73 mg/ml of sodium acetate trihydrate, 45 mg/ml of mannitol, 0.02 mg/ml of disodium EDTA dihydrate, 0.2 mg/ml of polysorbate 80, adjusted to pH 5.5 with glacial acetic acid, e.g. for intravenous use. In other embodiments, a formulation contains 50 mg/ml of antibody, 2.73 mg/ml of sodium acetate trihydrate, 45 mg/ml of mannitol, 0.02 mg/ml of disodium EDTA dihydrate, 0.4 mg/ml of polysorbate 80, adjusted to pH 5.5 with glacial acetic acid, e.g. for subcutaneous or intradermal use. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

In a preferred embodiment, the antibody is administered in a formulation as a sterile aqueous solution having a pH that ranges from about 5.0 to about 6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody, from about 1 millimolar to about 100 millimolar of histidine buffer, from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about 100 millimolar to about 400 millimolar of trehalose, and from about 0.01 millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

Another aspect of the present invention provides kits comprising an anti-MAdCAM antibody or antibody portion of the invention or a composition comprising such an antibody. A kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. A kit can also include instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method described below. In another preferred embodiment, the kit includes the antibody or a composition comprising it and one or more therapeutic agents that can be used in a method described below.

The nucleic acid molecules of the instant invention can be administered to a patient in need thereof via gene therapy. The therapy may be either in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both a heavy chain and a light chain are administered to a patient. In a more preferred embodiment, the nucleic acid molecules are administered such that they are stably integrated into chromosomes of B cells because these cells are specialized for producing antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and re-transplanted into a patient in need thereof. In another embodiment, precursor B cells or other cells are infected in viva using a recombinant virus known to infect the cell type of interest. Typical vectors used for gene therapy include liposomes, plasmids and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. After infection either in vivo or ex vivo, levels of antibody expression can be monitored by taking a sample from the treated patient and using any immunoassay known in the art or discussed herein.

In a preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain or an antigen-binding portion thereof of an anti-MAdCAM antibody and expressing the nucleic acid molecule. In another embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the light chain or an antigen-binding portion thereof of an anti-MAdCAM antibody and expressing the nucleic acid molecule. In a more preferred method, the gene therapy method comprises the steps of administering of an isolated nucleic acid molecule encoding the heavy chain or an antigen-binding portion thereof and an isolated nucleic acid molecule encoding the light chain or the antigen-binding portion thereof of an anti-MAdCAM antibody of the invention and expressing the nucleic acid molecules. The gene therapy method may also comprise the step of administering another anti-inflammatory or immunomodulatory agent.

Another aspect of the invention is a method of diagnosis of a condition where the antibody of the invention will be useful as a medicament, by testing whether abnormal binding of an antibody of the invention occurs in the patient. This can be done using various imaging techniques well known to the skilled person, such as x-ray analysis, gamma scintigraphy, magnetic resonance imaging (MRI), positron emission tomography or computed tomography (CT) and others.

One or more inhibitory anti-MAdCAM antibodies of the invention can be used as a vaccine or as adjuvants to a vaccine, and this is another aspect of the invention. In particular, because MAdCAM is expressed in lymphoid tissue, vaccine antigens can be advantageously targeted to lymphoid tissue by conjugating the antigen to an anti-MAdCAM antibody of the invention.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

A protein or polypeptide is “substantially pure,” “substantially homogeneous” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, even more preferably at least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptide that comprises a segment of at least 25 amino acids that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to MAdCAM under suitable binding conditions, (2) ability to inhibit α₄β₇ integrin and/or L-selectin binding to MAdCAM, or (3) ability to reduce MAdCAM cell surface expression in vitro or in vivo. Typically, polypeptide analogs comprise a conservative amino acid substitution (or insertion or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

An “immunoglobulin” is a tetrameric molecule. In a naturally-occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions to form an epitope-specific binding site. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917(1987); Chothia et al., Nature, 342:878-883(1989), each of which is incorporated herein by reference in their entirety.

The term “antibody” refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. In some embodiments, an antibody is an antigen-binding portion thereof. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)₂, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)₂ fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature, 341:544-546(1989)) consists of a VH domain. The term is also intended to include modified versions of the antibody such as pegylated antibodies or antibodies conjugated to detectable moieties such as enzymes (e.g. horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, fluorescent labels, and others.

As used herein, an antibody that is referred to as, e.g., 1.7.2, 1.8.2, 6.14.2, 6.34.2, 6.67.1, 6.77.2, 7.16.6, 7.20.5, 7.26.4 or 9.8.2, is a monoclonal antibody that is produced by the hybridoma of the same name. For example, antibody 1.7.2 is produced by hybridoma 1.7.2. An antibody that is referred to as 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod is a monoclonal antibody whose sequence has been modified from its corresponding parent by site-directed mutagenesis.

A single-chain antibody (scFv) is an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)). Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Hollinger, P., et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) and Poljak, R. J., et al., Structure, 2:1121-1123 (1994)). One or more CDRs from an antibody of the invention may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that specifically binds to MAdCAM. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispecific” or “bifunctional” antibody (diabody) has two different binding sites.

An “isolated antibody” is an antibody that (1) is not associated with naturally-associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species; or (4) does not occur in nature. Examples of isolated antibodies include an anti-MAdCAM antibody that has been affinity purified using MAdCAM, an anti-MAdCAM antibody that has been produced by a hybridoma or other cell line in vitro, and a human anti-MAdCAM antibody derived from a transgenic mammal or plant.

As used herein, the term “human antibody” means an antibody in which the variable and constant region sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g., to decrease possible immunogenicity, increase affinity, eliminate cysteines or glycosylation sites that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells which might impart glycosylation not typical of human cells. The term also emcompasses antibodies which have been raised in a transgenic mouse which comprises some or all of the human immunoglobulin heavy and light chain loci.

In one aspect, the invention provides a humanized antibody. In some embodiments, the humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans. In some embodiments, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293. In some embodiments, a humanized anti-MAdCAM antibody of the invention comprises the amino acid sequence of one or more framework regions of one or more human anti-MAdCAM antibodies of the invention.

In another aspect, the invention includes the use of a “chimeric antibody”. In some embodiments the chimeric antibody refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In a preferred embodiment, one or more of the CDRs are derived from a human anti-MAdCAM antibody of the invention. In a more preferred embodiment, all of the CDRs are derived from a human anti-MAdCAM antibody of the invention. In another preferred embodiment, the CDRs from more than one human anti-MAdCAM antibody of the invention are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human anti-MAdCAM antibody may be combined with CDR2 and CDR3 from the light chain of a second human anti-MAdCAM antibody, and the CDRs from the heavy chain may be derived from a third anti-MAdCAM antibody. Further, the framework regions may be derived from one of the same anti-MAdCAM antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.

A “neutralizing antibody,” “an inhibitory antibody” or antagonist antibody is an antibody that inhibits the binding of α₄β₇ or α₄β₇-expressing cells, or any other cognate ligand or cognate ligand-expressing cells, to MAdCAM by at least about 20%. In a preferred embodiment, the antibody reduces inhibits the binding of α₄β₇ integrin or α₄β₇-expressing cells to MAdCAM by at least 40%, more preferably by 60%, even more preferably by 80%, 85%, 90%, 95% or 100%. The binding reduction may be measured by any means known to one of ordinary skill in the art, for example, as measured in an in vitro competitive binding assay. An example of measuring the reduction in binding of α₄β₇-expressing cells to MAdCAM is presented in Example I.

Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art following the teachings of this specification. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known (Bowie et al., Science, 253:164 (1991)).

The term “k_(off)” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

The term “K_(d)” refers to the dissociation constant of a particular antibody-antigen interaction. An antibody is said to bind an antigen when the dissociation constant is ≦1 μM, preferably ≦100 nM and most preferably ≦10 nM.

The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearally along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077(1984); Stein et al., Nucl. Acids Res., 16:3209(1988); Zon et al., Anti-Cancer Drug Design 6:539(1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England(1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews, 90:543(1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

“Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

The term “selectively hybridize” referred to herein means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. “High stringency” or “highly stringent” conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. An example of “high stringency” or “highly stringent” conditions is a method of incubating a polynucleotide with another polynucleotide, wherein one polynucleotide may be affixed to a solid surface such as a membrane, in a hybridization buffer of 6×SSPE or SSC, 50% formamide, 5× Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA at a hybridization temperature of 42° C. for 12-16 hours, followed by twice washing at 55° C. using a wash buffer of 1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleotide sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.3, Accelrys, San Diego, Calif. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol., 183: 63-98 (1990); Pearson, Methods Mol. Biol., 132: 185-219 (2000); Pearson, Methods Enzymol., 266: 227-258 (1996); Pearson, J. Mol. Biol., 276: 71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleotide sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in Wisconsin Package Version 10.3, herein incorporated by reference.

A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

In the molecular biology art, researchers use the terms “percent sequence identity”, “percent sequence similarity” and “percent sequence homology” interchangeably. In this application, these terms shall have the same meaning with respect to nucleotide sequences only.

The term “substantial similarity” or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol., 24: 307-31 (1994), herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science, 256: 1443-45 (1992), herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Besffit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., Wisconsin package Version 10.3. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in Wisconsin package Version 10.3. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990); Pearson (2000)). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or labeled” refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ₁₁₁In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The following examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES Example 1 Identification of Potential Immunogenic Epitopes

CD4⁺ T (helper) cell epitopes are critical in driving T cell dependent immune responses to protein antigens. During the initiation of a T-cell dependent immune response dendritic cells capture, process and present protein antigen in the form of linear peptides bound in the groove of MHC class II. Binding of the T-cell receptor to the peptide/MHC-II complex by naïve CD4⁺ T cells can trigger a cascade in which T cells proliferate, differentiate and provide help by producing co-stimulatory cytokines (e.g. IL-4, IL-6) and by direct cell-cell contact (e.g. CD40) to B cells. The continued help from T cells is required during secondary B cell lymphopoiesis in which B cells rearrange their immunoglobulin chains to produce high affinity isotype switched immunoglobulin against the same antigen recognised by the T cells. It is the binding of the B cell produced immunoglobulins that can neutralise the effects of protein-based therapies. The identification of T cell epitopes contained within therapeutic antibodies is important in predicting immunogenicity in man. With their identification it may be desirable to engineer the MHC-II anchors out by site directed mutagenesis, generating non-immunogenic adducts that retain the therapeutic activity of the parent.

Six fully human anti-MAdCAM mAbs, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, 7.16.6 and 9.8.2 (previously described in WO2005/067620; hybridomas secreting 7.16.6 and 9.8.2 were deposited in the European Collection of Cell Cultures (ECACC), H.P.A at CAMR, Porton Down, Salisbury, Wiltshire SP4 0JG on 9 Sep. 2003 with the following deposit numbers: 7.16.6: Deposit No: 03090909; 9.8.2: Deposit No: 03090912), were subjected to an ex vivo assessment to identify potential T cell epitopes and determine their immunogenicity. Sequence overlapping peptides (15 mers of purity >90%) covering the framework (FR) and complementary determining regions (CDRs) of the heavy and kappa light chain counterparts of 6.22.2, 6.34.2, 6.67.1, 6.77.1, 7.16.6 and 9.8.2 were synthesised using standard methods. The peptides were screened against a panel of 40 human donors in multiple cultures of CD8⁺ T-cell depleted PMSC, providing a source of CD4⁺ cells and antigen presenting cells at physiological ratios. The 40 healthy donors were selected for screening on the basis of HLA-DR typing and represented >80% of the DR alleles expressed in the world's population. Individual peptides (1 and 5 μM) were screened in sextuplicate, in conjunction with two positive control peptides. Cells were treated with peptides for 7 days, then proliferation was measured by incorporation of ³H-thymidine (0.5 μCi/well; 18 hrs). The incorporation of radiolabel was determined by scintillation counting and expressed as a stimulation index (SI) as follows:

${SI} = \frac{{cpm}\mspace{14mu} {of}\mspace{14mu} {test}\mspace{14mu} {peptide}}{{Cpm}\mspace{14mu} {of}\mspace{14mu} {untreated}\mspace{14mu} {control}}$

A T-cell epitope is defined as a peptide giving a SI >2 in two or more independent donors.

In addition to the peptide-induced proliferation response, peptides were tested on PBMC donors in an ELISPOT assay. This method captures released cytokines and enables quantification of activated cells within the whole population. The release of IL-2, the principle cytokine secreted by activated CD4⁺ T cells, was selected to identify T-cell stimulated by the peptide epitope. As with the proliferation assay, donor samples were incubated with peptide for 7 days prior to assessment. On analysis each stimulated T cell was represented as a spot of IL-2 release. For each donor and peptide (1 and 5 μM) combination the number of spots/well were determined and a stimulation index calculated as a ratio:

${SI} = \frac{{spots}\text{/}{well}\mspace{14mu} {of}\mspace{14mu} {{tes}t}\mspace{14mu} {peptide}}{{spots}\text{/}{well}\mspace{14mu} {of}\mspace{14mu} {untreated}\mspace{14mu} {control}}$

In general there should be good overlap in the responses observed between the proliferation and ELISPOT assays, though differences in the kinetics and magnitude of responses between the two assays can lead to some lack of correlation.

Table 1 describes the data from the T-cell proliferation and ELISPOT assays. Based on these analyses, T cell epitopes were identified in FR2/CDR2 region of the 6.22.2-mod heavy chain; CDR2, FR3/CDR3 and CDR3 of the kappa light chain of 6.34.2-mod light chain; FR3/CDR3 of the 6.67.1-mod kappa light chain; CDR3 of the 6.77.1-mod heavy chain; CDR2 of the 7.16.6 heavy chain and FR3/CDR3 of the 7.16.6 kappa light chain; and FR3/CDR3 of the 9.8.2 heavy chain.

TABLE 1 The induction of T cell proliferation and IL-2 production in PBMCs from 40 healthy volunteers incubated with different peptides derived from the anti-MAdCAM mAbs 6.22.2-mod, 6.34.2-mod, 6.67.1- mod, 6.77.1-mod, 7.16.6 and 9.8.2. The sequences of each peptide are indicated, residues which differ from the germline sequence are in bold. Table 1: Prolif- eration ELIISPOT Antibody Chain Sequence (SI >2) (SI >2) 6.22.2 Heavy SGFTFSSDGMHWVRQ 1 (SEQ ID NO: 39) EWVAIIWYDGSNKYY 2 Light RASQRIGSSLHWYQQ 1 6.34.2 Heavy AVISNDGNNKYYADS 1 Light ISSYLNWFQQKPGKA 1 YLNWFQQKPGKAPKL 1 1 APKLLIYAASGLKRG 3 1 LLIYAASGLKRGVPS 1 SGLKRGVPSRFSGSG 1 QPEDFATYYCHQSYS 1 DFATYYCHQSYSLPF 2 CHQSYSLPFTFGPGT 1 3 SYSLPFTFGPGTKVD 1 6.67.1 Heavy GRIYTSGGTNSNPSL 1 RDRITIIRGLIPSFF 1 ITIIRGLIPSFFDYW 1 LIPSFFDYWGQGTLV 1 Light PPKLLIYWASIREYG 1 AEDVAVYFCQQYYSI 1 VAVYFCQQYYSIPPL 3 YFCQQYYSIPPLTFG 1 YSIPPLTFGGGTKVE 1 6.77.1 Heavy AVYYCARDGYSSQWS 1 RDGYSSGWSYYYYYG 1 YSSGWSYYYYYGMDV 3 GWSYYYYYGMDVWGO 4 Light ISCKSSQSLLLSDGK 1 EAEDVGVYSCMQSIQ 1 VYSCMQSIQLMSSFG 1 SIQLMSSFGQGTKLE 1 1 7.16.6 Heavy SVYSGNTNYAQKVQG 2 AVYYCAREGSSSSGD 1 SGDYYYGMDVWGQGT 1 Light VEAEDVGIYYCMQNI 1 EDVGIYYCMQNIQLP 4 4 GIYYCMQNIQLPWTF 1 1 9.8.2 heavy VAVIWYDGSNEYYAD 1 IWYDGSNEYYADSVK 1 1 AEDTAVYYCARGAYH 1 1 TAVYYCARGAYHFAY 3 ARGAYHFAYWGQGTL 1 Light SLQPEDIATYSCQHS 1 PEDIATYSCQHSDNL 1 IATYSCQHSDNLTFG 1 YSCQHSDNLTFGQGT 1

Example 2 Characterisation of Modified Variants of 7.16.6

The T-cell proliferation and EL1SPOT analysis identified a number of potential T-cell epitopes in the MAdCAM mAbs 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, 7.16.6 and 9.8.2. The major T-cell epitope in the kappa light chain of 7.16.6 was reverted to germline by site directed mutagenesis (QuikChange, Stratagene) according to manufacturers instructions. The sequences of the oligonucleotides used to generate the modified forms are indicated below: primer pairs 7.16.6_V5 and 7.16.6_V3 were used to generate the 7.16.6_V adduct (VH=wild type; VL=190V); 7.16.6_S5 and 7.16.6_S3 were used to generate the 7.16.6_S adduct (VH=wild type; VL=N96S); 7.16.6_VS5 and 7.16.6_VS3 were used to generate the 7.16.6_VS adduct (VH=wild type; VL=I90V, N96S). The pEE12.1 vector containing the 7.16.6 heavy chain cDNA (described in WO2005/067620). Briefly, a functional eukaryotic expression vector containing the respective heavy and light chain sequences were assembled as follows: The heavy chain cDNA inserts were excised from the required pEE6.1 with NotI/SalI, and the purified fragments were cloned into identical sites into the corresponding pEE12.1 vector containing the required versions of the kappa light chain sequences.) was used as a template for the QuikChange PCR.

7.16.6_V5 (SEQ ID NO: 84) GGCTGAGGATGTTGGGGTTTATTACTGCATGC 7.16.6_V3 (SEQ ID NO: 85) GCATGCAGTAATAAACCCCAACATCCTCAGCC 7.16.6_S5 (SEQ ID NO: 86) CTGCATGCAAAGTATACAGCTTCCGTGGAC 7.16.6_S3 (SEQ ID NO: 87) GTCCACGGAAGCTGTATACTTTGCATGCAG 7.16.6_VS5 (SEQ ID NO: 88) GGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCG 7.16.6_VS3 (SEQ ID NO: 89) CGGAAGCTGTATACTTTGCATGCAGTAATAAACCCCAACATCC

pEE12.1 constructs containing the modified kappa light chains of 7.16.6_V, 7.16.6_S and 7.16.6_VS along with the parent 7.16.6 heavy chain were generated from the QuikChange PCR products and fully sequence verified. A similar PCR approach was used to generate the 7.16.6_L variant (VH=V64L, VL=wild type), using the following oligonucleotides and QuikChange PCR:

7.16.6_L5 CTATGCACAGAAGCTTCAGGGCAGAGTCAC (SEQ ID NO: 90) 7.16.6_L3 GTGACTCTGCCCTGAAGCTTCTGTGCATAG (SEQ ID NO: 91)

Parent kappa light chain and 7.16.6_VS variant were used to generate the 7.16.6_L and 7.16.6_LVS (VH=V64L; VL=I90V, N965) variant as described in WO2005/067620. Recombinant material was purified by affinity chromatography from FreeStyle HEK293 cells (Invitrogen) transiently transfected with the appropriate construct.

The purified antibodies were assessed for activity in an adhesion assay using MAdCAM-IgG₁ Fc fusion protein:

An EcoRI/BglII cDNA fragment encoding the mature extracellular, immunoglobulin-like domain of MAdCAM was excised from a pINCY Incyte clone (3279276) and cloned into EcoRI/BamHI sites of the pIG1 vector (Simmons, D. L. (1993) in Cellular Interactions in Development: A Practical Approach, ed. Hartley, D. A. (Oxford Univ. Press, Oxford), pp. 93-127.)) to generate an in frame IgG₁ Fc fusion. The resulting insert was excised with EcoRI/NotI and cloned into pCDNA3.1+ (Invitrogen). The MAdCAM-IgG₁ Fe cDNA in the vector was sequence confirmed. The amino acid sequence of the MAdCAM-IgG₁ Fc fusion protein is shown below:

(SEQ ID NO: 92) MDFGLALLLAGLLGLLLG QSLQVKPLQVEPPEPVVAVALGASRQLTCRLA CADRGAVVQWRGLDTSLGAVQSDTGRSVLTVRNASLSAAGTRVCVGSCGG RTFQHTVQLLVYAFPDQLTVSPAALVPGDPEVACTAHKVTPVDPNALSFS LLVGGQELEGAQALGPEVQEEEEEPQGDEDVLFRVTERWRLPPLGTPVPP ALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDTTSPESPD TTSQEPPDTTSQEPPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRS PGSTRTRRPEIQPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Underlined: signal peptide Bold: MAdCAM extracellular domain

CHO-DHFR cells were transfected with pCDNA3.1+ vector containing MAdCAM-IgG₁ Fc fusion protein cDNA and stable clones expressing MAdCAM-IgG₁ Fc fusion protein selected in Iscove's media containing 600 μg/mL G418 and 100 ng/mL methotrexate. For protein expression, a hollow fibre bioreactor was seeded with stably expressing MAdCAM-IgG₁ Fc CHO cells in Iscove's media containing 10% low IgG fetal bovine serum (Gibco), non essential amino acids (Gibco), 2 mM glutamine (Gibco), sodium pyruvate (Gibco), 100 μg/mL G418 and 100 ng/mL methotrexate, and used to generate concentrated media supernatant. The MAdCAM-IgG₁ Fc fusion protein was purified from the harvested supernatant by affinity chromatography. Briefly, supernatant was applied to a HiTrap Protein G Sepharose (5 mL, Pharmacia) column (2 mL/min), washed with 25 mM Tris pH 8, 150 mM NaCl (5 column volumes) and eluted with 100 mM glycine pH 2.5 (1 mL/min), immediately neutralising fractions to pH 7.5 with 1M Tris pH 8. Fractions containing MAdCAM-IgG₁ Fc fusion protein were identified by SDS-PAGE, pooled together and applied to a Sephacryl S100 column (Pharmacia), pre-equilibrated with 35 mM BisTris pH 6.5, 150 mM NaCl. The gel filtration was performed at 0.35 mL/min, collecting a peak of MAdCAM-IgG₁ Fc fusion protein in ca. 3×5 mL fractions. These samples were pooled and applied to a Resource Q (6 mL, Pharmacia) column, pre-equilibrated in 35 mM BisTris pH6.5. The column was washed with 5 column volumes of 35 mM Bis Tris pH 6.5, 150 mM NaCl (6 mL/min) and MAdCAM-IgG₁ Fc fusion protein eluted into a 4-6 mL fraction with 35 mM Bis Tris pH 6.5, 400 mM NaCl. At this stage the protein was 90% pure and migrating as a single band at approximately 68 kD by SDS-PAGE. For use as an immunogen and all subsequent assays, the material was buffer exchanged into 25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 50% glycerol and stored as aliquots at −80° C.

100 μl of a 4.5 μg/mL solution of purified MAdCAM-IgG₁ Fc fusion protein in Dulbecco's PBS was adsorbed to 96 well Black Microfluor “B” u-bottom (Dynex #7805) plates overnight at 4° C. The MAdCAM coated plates were then inverted and excess liquid blotted off, prior to blocking at 37° C. for at least 1 hour in 10% BSN PBS. During this time cultured JY cells were counted using tryptan blue exclusion (should be approximately 8×10⁵ cells/mL) and 20×10⁶ cells/assay plate pipetted into a 50 mL centrifuge tube. JY cells were cultured in RPM11640 media (Gibco), containing 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (Life Technologies #10108-165) and seeded at 1-2×10⁵/mL every 2-3 days to prevent the culture from differentiating. The cells were washed twice with RPMI 1640 media (Gibco) containing 2 mM L-glutamine (Gibco) by centrifugation (240 g), resuspending the final cell pellet at 2×10⁶ cells/mL in RPMI 1640 for Calcein AM loading. Calcein AM (Molecular Probes #C-3099) was added to the cells as a 1:200 dilution in DMSO (ca. final concentration 5 μM) and the cells protected from light during the course of the incubation (37° C. for 30 min). During this cell incubation step the antibodies to be tested, were diluted as follows: for single dose testing, the antibodies were made up to 3 μg/mL (1 μg/mL final) in 0.1 mg/mL BSA (Sigma #A3059) in PBS; for full IC₅₀ curves, the antibodies were diluted in 0.1 mg/mL BSA/PBS, with 3 μg/mL (1 μg/mL final) being the top concentration, then doubling dilutions (1:2 ratio) across the plate. The final well of the row was used for determining total binding, so 0.1 mg/ml BSA in PBS was used.

After blocking, the plate contents were flicked out and 50 μL of antibodies/controls were added to each well and the plate incubated at 37° C. for 20 min. During this time, Calcein-loaded JY cells were washed once with RPMI 1640 media containing 10% fetal bovine serum and once with 1 mg/mL BSA/PBS by centrifugation, resuspending the final cell pellet to 1×10⁶/mL in 1 mg/mL BSA/PBS. 100 μL of cells were added to each well of the U bottomed plate, the plate sealed, briefly centrifuged (1000 rpm for 2 min) and the plate then incubated at 37° C. for 45 min. At the end of this time, the plates were washed with a Skatron plate washer and fluorescence measured using a Wallac Victor² 1420 Multilabel Reader (excitation λ485 nm, emission λ535 nm count from top, 8 mm from bottom of plate, for 0.1 sec with normal emission aperture). For each antibody concentration, percent adhesion was expressed as a percentage of maximal fluorescence response in the absence of any antibody minus fluorescence associated with non-specific binding. The IC₅₀ value is defined as the anti-MAdCAM antibody concentration at which the adhesion response is decreased to 50% of the response in the absence of anti-MAdCAM antibody. Antibodies that were able to inhibit the binding of JY cells to MAdCAM-IgG₁ Fc fusion with an IC₅₀ value <0.1 μg/mL, were considered to have potent antagonist activity and were progressed to the MAdCAM-CHO adhesion assay.

The IC₅₀ values are summarised in Table 2 (all datapoints given in μg/mL):

TABLE 2 Kd, k_(a), and k_(d) values for the interaction of MAdCAM with the anti-MAdCAM antibody 7.16.6 and 5 clones derived from it. Kd k_(a) k_(d) IC₅₀ (pM) (M⁻¹ s⁻¹) (s⁻¹) (ng/mL) s.d n 7.16.6 7.3  3.3 × 10⁵  2.42 × 10⁻⁶* 24.7 7.6 6 7.16.6_S 23.2 1.83 × 10⁵  4.27 × 10⁻⁶* 34.2 7.4 5 7.16.6_V 162 1.53 × 10⁵ 2.49 × 10⁻⁵ 41.2 20.9 5 7.16.6_VS 90.8 2.40 × 10⁵ 2.18 × 10⁻⁵ 58.2 51.2 5 7.16.6_L 79.5 1.52 × 10⁵ 1.21 × 10⁻⁵ 35.6 10 5 7.16.6_LVS 119 1.94 × 10⁵ 2.31 × 10⁻⁵ 45.8 16.4 5 *Indicates that these off rate values are just below the lower limit (5 × 10⁻⁶ s⁻¹) for BIAcore. The affinity (KD) values should therefore more accurately be quoted as: <15 pM (=5 × 10⁻⁶/3.3 × 10⁵) for 7.16.6 and <27.3 pM (=5 × 10⁻⁶/1.83 × 10⁵) for 7.16.6_V The off rates for the other four antibodies are within the limits of the instrument.

The results show that the variants still bind MAdCAM with high affinity, sufficiently high for therapeutic use.

7.16.6, 7.16.6_V, 7.16.6_S, 7.16.6_VS, 7.16.6_L and 7.16.6_LVS also bound to CHO cells expressing MAdCAM, as detected by flow cytometry.

The Kd values shown in Table 2 were determined by BIAcore, essentially as described in Example II in WO 2005/067620, with the following modifications:

Sample Injections

Flow cells used: Either 2 with 1 as a reference, or 4 with 3 as a reference

Flow rate: 100 μL/min

Injection time: 2 minutes

Wait after injection: 60 minutes(=dissociation time)

Injection cycles: Triplicate of all concentrations, plus six injections of buffer blank

Regeneration Method

Solution/flow rate/injection time:

Regeneration Immobilised Regeneration flow rate, Injection time antibody solution μL/min (vol) 7.16.6 50 mM Phosphoric 50 12 seconds Acid (10 μL) 7.16.6_V 60 mM Phosphoric 50 54 seconds Acid (45 μL) 7.16.6_S 4M Magnesium 100 15 seconds Chloride (25 μL) 7.16.6_VS 50 mM Phosphoric 50 12 seconds Acid (10 μL) 7.16.6_L 7.5 mM Sodium 50  6 seconds Hydroxide   (5 μL) 7.16.6_LVS 5 mM Sodium 50  6 seconds Hydroxide   (5 μL)

Stabilisation time after regeneration: 5 minutes

Example 3 Immunohistochemistry Staining of Cynomolaus Tissues with Variants of 7.16.6

Immunohistochemistry was essentially performed as described in Example III in WO 2005/067620. The results are summarized in Table 3 below.

TABLE 3 Summary of staining of normal cynomolgus tissues with anti-hMAdCAM 1/500 dilution (ca. 0.3 μg/mL), 1 hour incubation at room temperature). 7.16.6_LVS 7.16.6_L 7.16.6_(—) 17.16.6_S 7.16.6_VS Neg. Control Tissue Positive staining Ileum/Peyer's 2-3+ HEV (PP), Vasc. = > = = = <<< Patch (PP) endothelium (lamina propria- LP) Cecum 3+ vasc. endothelium = = = = = <<< (LP) Spleen 2-4+ Sinus-lining cells < = = < < <<< (marginal zone of white pulp) Mesenteric 4+ HEV = = = = = <<< LN Axillary LN trace Dendritic-like = = = = = <<< cells around lymphoid follicles Pancreas 1+ Vas endothelium = = < = = <<< (venules of exocrine lobules) Negative Staining Kidney* 0  — = = = = = = Heart 0  — = = = = = = (ventricle) Thyroid 0  — = = = = = = Strong staining in tubules attributable to non-specific binding of the secondary antibody and interpreted as background noise.

Example 4 Characterisation of Modified Variants of 6.22.2. 6.34.2. 6.67.1. 6.77.1 and 9.8.2

Using similar methods as described here in Example 2 and referred to in WO2005/067620, some of the significant potential T cell epitopes in 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 9.8.2 described in Table 1 were modified to germline by substitution or deletion to generate purified recombinant 6.22.2-mod_V (SEQ ID No. 2), 6.34.2_mod_SSQ (SEQ ID No. 8), 6.67.1-mod_Y (SEQ ID No. 14), 6.77.1-mod_ΔAS (SEQ ID No. 22) and 9.8.2-mod_ΔRGAYH,D (SEQ ID No. 36) chain variants. The IC₅₀ potencies of the following mAbs referred to as 6.22.2-mod_V (SEQ ID Nos. 2 and 4), 6.34.2_mod_SSQ (SEQ ID Nos. 6 and 8), 6.67.1-mod_Y (SEQ ID Nos. 14 and 20), 6.77.1-mod_ΔAS (SEQ ID Nos. 22 and 24) and 9.8.2-mod_ΔRGAYH,D (SEQ ID Nos. 36 and 38) in the MAdCAM-IgG₁ Fc fusion: JY cell binding assay are represented in Table 4 compared with their parental counterpart 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 9.8.2 mAbs. All modified variants maintained similar antagonist potency to their parental mAbs.

TABLE 4 Comparison of IC₅₀ potencies of the 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 9.8.2 modified mAbs, 6.22.2-mod_V, 6.34.2_mod_SSQ, 6.67.1-mod_Y, 6.77.1-mod_ΔAS and 9.8.2-mod_ΔRGAYH,D respectively in the MAdCAM-IgG₁ Fc fusion: JY cell binding assay IC₅₀ (μg/mL) S.D n IC₅₀ (μg/mL) S.D n 6.22.2 0.018 0.0109 4 6.22.2-mod_V 0.076 0.033 7 6.34.2 0.013 0.0079 4 6.34.2-mod_SSQ 0.086 0.04 11 6.67.1 0.013 0.0071 4 6.67.1-mod_Y 0.063 0.025 6 6.77.1 0.022 0.0045 4 6.77.1-mod_ΔAS 0.0485 0.0135 7 9.8.2 0.02 0.0047 4 9.8.2_ΔRGAYH,D 0.072 0.03 6

Sequences:

The sequences of the signal peptide sequence (or the bases encoding the same) are indicated in lower case and underlined. The variable regions are in upper case. The constant regions are in upper case and underlined in the protein sequences. Bold are residues modified from the originally disclosed sequences (WO2005/067620).

SEQ ID NO: 1 6.22.2-mod_V Heavy Chain Nucleotide Sequence    1 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt   51 ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG  101 GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC  151 GATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT  201 GGCAGTGATA TGGTATGATG GAAGTAATAA ATATTATGCA GACTCCGTGA  251 AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG  301 CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTATATT ACTGTGCGAG  351 AGATCCCGGC TACTATTACG GTATGGACGT CTGGGGCCAA GGGACCACGG  401 TCACCGTCTC CTCAGCTTCC ACCAAGGGCC CATCCGTCTT CCCCCTGGCG  451 CCCTGCTCTA GAAGCACCTC CGAGAGCACA GCGGCCCTGG GCTGCCTGGT  501 CAAGGACTAC TTCCCCGAAC CGGTGACGGT GTCGTGGAAC TCAGGCGCTC  551 TGACCAGCGG CGTGCACACC TTCCCAGCTG TCCTACAGTC CTCAGGACTC  601 TACTCCCTCA GCAGCGTGGT GACCGTGCCC TCCAGCAACT TCGGCACCCA  651 GACCTACACC TGCAACGTAG ATCACAAGCC CAGCAACACC AAGGTGGACA  701 AGACAGTTGA GCGCAAATGT TGTGTCGAGT GCCCACCGTG CCCAGCACCA  751 CCTGTGGCAG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC  801 CCTCATGATC TCCCGGACCC CTGAGGTCAC GTGCGTGGTG GTGGACGTGA  851 GCCACGAAGA CCCCGAGGTC CAGTTCAACT GGTACGTGGA CGGCGTGGAG  901 GTGCATAATG CCAAGACAAA GCCACGGGAG GAGCAGTTCA ACAGCACGTT  951 CCGTGTGGTC AGCGTCCTCA CCGTTGTGCA CCAGGACTGG CTGAACGGCA 1001 AGGAGTACAA GTGCAAGGTC TCCAACAAAG GCCTCCCAGC CCCCATCGAG 1051 AAAACCATCT CCAAGACAAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC 1101 CCTGCCCCCA TCCCGGGAGG AGATGACCAA GAACCAGGTC AGCCTGACCT 1151 GCCTGGTCAA AGGCTTCTAC CCCAGCGACA TCGCCGTGGA GTGGGAGAGC 1201 AATGGGCAGC CGGAGAACAA CTACAAGACC ACACCTCCCA TGCTGGACTC 1251 CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC AAGAGCAGGT 1301 GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 1351 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATGATAG SEQ ID NO: 2 6.22.2-mod_V Predicted Heavy Chain Protein Sequence    1 mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS   51 DGMHWVRQAP GKGLEWVAVI WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL  101 QMNSLRAEDT AVYYCARDPG YYYGMDVWGQ GTTVTVSSAS TKGPSVFPLA  151 PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL  201 YSLSSVVTVP SSNFGTQTYT CNVDHKPSNT KVDKTVERKC CVECPPCPAP  251 PVAGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE  301 VHNAKTKPRE EQFNSTFRVV SVLTVVHQDW LNGKEYKCKV SNKGLPAPIE  351 KTISKTKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES  401 NGQPENNYKT TPPMLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH  451 NHYTQKSLSL SPGK SEQ ID NO: 3 6.22.2-mod Kappa Light Chain Nucleotide Sequence    1 atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagcttc   51 caggggtGAA ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC  101 CAAAAGAGAA AGTCACCATC ACCTGCCGGG CCAGTCAGAG AATTGGTAGT  151 AGCTTACACT GGTACCAGCA GAAACCAGAT CAGTCTCCAA AACTCCTCAT  201 CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG TTCAGTGGCA  251 GTGGATCTGG GACAGATTTC ACCCTCACCA TCAATAGCCT GGAAGCTGAA  301 GATGCTGCAA CTTATTACTG TCATCAGAGT GGTCGTTTAC CGCTCACTTT  351 CGGCGGAGGG ACCAAGGTGG AGATCAAACG AACTGTGGCT GCACCATCTG  401 TCTTCATCTT CCCGCCATCT GATGAGCAGT TGGAGTCTGG AACTGCCTCT  451 GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG  501 GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG  551 AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG  601 AGCAAAGCAG ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA  651 TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT  701 AGTGA SEQ ID NO: 4 6.22.2-mod Predicted Kappa Light Chain Amino Acid Sequence    1 mlpsqligfl llwvpasrgE IVLTQSPDFQ SVTPKEKVTI TCRASQRIGS   51 SLHWYQQKPD QSPKLLIKYA SQSFSGVPSR FSGSGSGTDF TLTINSLEAE  101 DAATYYCHQS GRLPLTFGGG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS  151 VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL  201 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC SEQ ID NO. 5 6.34.2-mod Heavy Chain Nucleotide Sequence    1 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt   51 ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG  101 GGAGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC  151 TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT  201 GGCAGTTATA TCAAATGATG GAAATAATAA ATACTATGCA GACTCCGTGA  251 AGGGCCGATT CACCATCTCC AGAGACAATT CCAAAAACAC GCTGTATCTG  301 CAAATGAACA GCCTGCGCGC TGAGGACACG GCTGTGTATT ACTGTGCGAG  351 AGATAGTACG GCGATAACCT ACTACTACTA CGGAATGGAC GTCTGGGGCC  401 AAGGGACCAC GGTCACCGTC TCCTCAGCTT CCACCAAGGG CCCATCCGTC  451 TTCCCCCTGG CGCCCTGCTC TAGAAGCACC TCCGAGAGCA CAGCGGCCCT  501 GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA  551 ACTCAGGCGC TCTGACCAGC GGCGTGCACA CCTTCCCAGC TGTCCTACAG  601 TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAA  651 CTTCGGCACC CAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA  701 CCAAGGTGGA CAAGACAGTT GAGCGCAAAT GTTGTGTCGA GTGCCCACCG  751 TGCCCAGCAC CACCTGTGGC AGGACCGICA GTCTTCCTCT TCCCCCCAAA  801 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG  851 TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA CTGGTACGTG  901 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG AGGAGCAGTT  951 CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT 1001 GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGGCCTCCCA 1051 GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC CCCGAGAACC 1101 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG 1151 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ACCCCAGCGA CATCGCCGTG 1201 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC 1251 CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG 1301 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT 1351 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG 1401 TAAATGATAG SEQ ID NO. 6 6.34.2-mod Predicted Heavy Chain Amino Acid Sequence    1 mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS   51 YGMHWVRQAP GKGLEWVAVI SNDGNNKYYA DSVKGRFTIS RDNSKNTLYL  101 QMNSLRAEDT AVYYCARDST AITYYYYGMD VWGQGTTVTV SSASTKGPSV  151 FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ  201 SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP  251 CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV  301 DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP  351 APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV  401 EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH  451 EALHNHYTQK SLSLSPGK SEQ ID NO: 7 6.34.2-mod_SSQ Kappa Light Chain Nucleotide Sequence    1 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct   51 ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT  101 CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAGT  151 ATTAGTAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA  201 GCTCCTGATC TATGCTGCAT CCAGTTTGAG TCAGGGGGTC CCATCACGGT  251 TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGTTCTCTG  301 CAACCTGAGG ATTTTGCAAC TTACTACTGT CACCAGAGTT ACAGTCTCCC  351 ATTCACTTTC GGCCCTGGGA CCAAGGTGGA TATCAAACGA ACTGTGGCTG  401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA  451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA  501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA  551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC  601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA  651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG  701 GAGAGTGTTA GTGA SEQ ID NO: 8 6.34.2-mod_SSQ Predicted Kappa Light Chain Protein Sequence    1 mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQS   51 ISSYLNWYQQ KPGKAPKLLI YAASSLSQGV PSRFSGSGSG TDFTLTISSL  101 QPEDFATYYC HQSYSLPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG  151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST  201 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO: 9 6.34.2-mod_QT Kappa Light Chain Nucleotide Sequence    1 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct   51 ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT  101 CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAGT  151 ATTAGTAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA  201 GCTCCTGATC TATGCTGCAT CCGGTTTGAA GCGTGGGGTC CCATCACGGT  251 TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGTTCTCTG  301 CAACCTGAGG ATTTTGCAAC TTACTACTGT CAGCAGAGTT ACAGTACTCC  351 ATTCACTTTC GGCCCTGGGA CCAAAGTGGA TATCAAACGA ACTGTGGCTG  401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA  451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA  501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA  551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC  601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA  651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG  701 GAGAGTGTTA GTGA SEQ ID NO: 10 6.34.2-mod_QT Predicted Kappa Light Chain Protein Sequence    1 mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQS   51 ISSYLNWYQQ KPGKAPKLLI YAASGLKRGV PSRFSGSGSG TDFTLTISSL  101 QPEDFATYYC QQSYSTPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG  151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST  201 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO: 11 6.34.2-mod_SSQ, QT Kappa Light Chain Nucleotide Sequence    1 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct   51 ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT  101 CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAGT  151 ATTAGTAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA  201 GCTCCTGATC TATGCTGCAT CCAGTTTGAG TCAGGGGGTC CCATCACGGT  251 TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGTTCTCTG  301 CAACCTGAGG ATTTTGCAAC TTACTACTGT CAGCAGAGTT ACAGTACTCC  351 ATTCACTTTC GGCCCTGGGA CCAAAGTGGA TATCAAACGA ACTGTGGCTG  401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA  451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA  501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA  551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC  601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA  651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG  701 GAGAGTGTTA GTGA SEQ ID NO: 12 6.34.2-mod_SSQ, QT Predicted Kappa Light Chain Protein Sequence    1 mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQS   51 ISSYLNWYQQ KPGKAPKLLI YAASSLSQGV PSRFSGSGSG TDFTLTISSL  101 QPEDFATYYC QQSYSTPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG  151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST  201 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO:13 6.67.1-mod Heavy Chain Nucleotide Sequence    1 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt   51 cctgtccCAG GTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT  101 CGGAGACCCT GTCCCTCACC TGCACTGTCT CTGGTGACTC CATCAGTAGT  151 AACTATTGGA GCTGGATCCG GCAGCCCGCC GGGAAGGGAC TGGAGTGGAT  201 TGGGCGTATC TATACCAGTG GGGGCACCAA CTCCAACCCC TCCCTCAGGG  251 GTCGAGTCAC CATGTCAGTA GACACGTCCA AGAACCAGTT CTCTCTGAAA  301 CTGAGTTCTG TGACCGCCGC GGACACGGCC GTGTATTACT GTGCGAGAGA  351 TCGTATTACT ATAATTCGGG GACTTATTCC ATCCTTCTTT GACTACTGGG  401 GCCAGGGAAC CCTGGTCACC GTCTCCTCAG CTTCCACCAA GGGCCCATCC  451 GTCTTCCCCC TGGCGCCCTG CTCTAGAAGC ACCTCCGAGA GCACAGCGGC  501 CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT  551 GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA  601 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG  651 CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA  701 ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA  751 CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC  801 AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG  851 TGGTGGTGGA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC  901 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA  951 GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001 ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101 ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251 TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401 GGGTAAATGA TAG SEQ ID NO. 14 6.67.1-mod Predicted Heavy Chain Amino Acid Sequence    1 mkhlwffLLL vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGDSISS   51 NYWSWIRQPA GKGLEWIGRI YTSGGTNSNP SLRGRVTMSV DTSKNQFSLK  101 LSSVTAADTA VYYCARDRIT IIRGLIPSFF DYWGQGTLVT VSSASTKGPS  151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL  201 QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP  251 PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY  301 VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL  351 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA  401 VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM  451 HEALHNHYTQ KSLSLSPGK SEQ ID NO. 15 6.67.1-mod_Y Kappa Light Chain Nucleotide Sequence    1 atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg   51 tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT  101 CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA  151 TACAGCTCCA ACAATAAGAA CTACTTAGCT TGGTACCAAC AGAAACCAGG  201 ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG  251 TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC  301 ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTATT GTCAACAATA  351 TTATAGTATT CCTCCCCTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA  401 AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG  451 CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA  501 TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG  551 GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC  601 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA  651 AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA  701 AGAGCTTCAA CAGGGGAGAG TGTTAGTGA SEQ ID NO: 16 6.67.1-mod_Y Predicted Kappa Light Chain Amino Acid Sequence    1 mvlqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL   51 YSSNNKNYLA WYQQKPGQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT  101 ISSLQAEDVA VYYCQQYYSI PPLTFGGGTK VEIKRTVAAP SVFIFPPSDE  151 QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY  201 SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C SEQ ID NO: 17 6.67.1-mod_TΔP Kappa Light Chain Nucleotide Sequence    1 atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg   51 tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT  101 CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA  151 TACAGCTCCA ACAATAAGAA CTACTTAGCT TGGTACCAAC AGAAACCAGG  201 ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG  251 TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC  301 ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTTCT GTCAACAATA  351 TTATAGTACC CCT---CTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA  401 AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG  451 CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA  501 TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG  551 GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC  601 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA  651 AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA  701 AGAGCTTCAA CAGGGGAGAG TGTTAGTGA SEQ ID NO: 18 6.67.1-mod_TΔP Predicted Kappa Light Chain Amino Acid Sequence    1 mvlqtqyfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL   51 YSSNNKNYLA WYQQKPGQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT  101 ISSLQAEDVA VYFCQQYYST P-LTFGGGTK VEIKRTVAAP SVFIFPPSDE  151 QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY  201 SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C SEQ ID NO: 19 6.67.1-mod_Y, TΔP Kappa Light Chain Nucleotide Sequence    1 atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg   51 tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT  101 CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA  151 TACAGCTCCA ACAATAAGAA CTACTTAGCT TGGTACCAAC AGAAACCAGG  201 ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG  251 TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC  301 ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTATT GTCAACAATA  351 TTATAGTACC CCT---CTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA  401 AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG  451 CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA  501 TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG  551 GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC  601 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA  651 AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA  701 AGAGCTTCAA CAGGGGAGAG TGTTAGTGA SEQ ID NO: 20 6.67.1-mod_Y, TΔP Predicted Kappa Light Chain Amino Acid Sequence    1 mvlqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL   51 YSSNNKNYLA WYQQKPGQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT  101 ISSLQAEDVA VYYCQQYYST P-LTFGGGTK VEIKRTVAAP SVFIFPPSDE  151 QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY  201 SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C SEQ ID NO: 21 6.77.1-mod_ΔS Heavy Chain Nucleotide Sequence    1 atggaactgg ggctccgctg ggttttcctt gttgctattt tagaaggtgt   51 ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCCTG GTCAAGCCTG  101 GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC  151 TATAGCATGA ACTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT  201 CTCATCCATT AGTAGTAGTA GTAGTTACAT ATACTACGCA GACTCAGTGA  251 AGGGCCGATT CACCATCTCC AGAGACAACG CCAAGAACTC ACTGTATCTG  301 CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG  351 AGATGGGTAT AGCAGTGGCT GG---TACTA CTACTACTAC GGTATGGACG  401 TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGCTTC CACCAAGGGC  451 CCATCCGTCT TCCCCCTGGC GCCCTGCTCT AGAAGCACCT CCGAGAGCAC  501 AGCGGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG  551 TGTCGTGGAA CTCAGGCGCT CTGACCAGCG GCGTGCACAC CTTCCCAGCT  601 GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC  651 CTCCAGCAAC TTCGGCACCC AGACCTACAC CTGCAACGTA GATCACAAGC  701 CCAGCAACAC CAAGGTGGAC AAGACAGTTG AGCGCAAATG TTGTGTCGAG  751 TGCCCACCGT GCCCAGCACC ACCTGTGGCA GGACCGTCAG TCTTCCTCTT  801 CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA  851 CGTGCGTGGT GGTGGACGTG AGCCACGAAG ACCCCGAGGT CCAGTTCAAC  901 TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCACGGGA  951 GGAGCAGTTC AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC 1001 ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 1051 GGCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC 1101 CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA 1151 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC 1201 ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC 1251 CACACCTCCC ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC 1301 TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 1351 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT 1401 GTCTCCGGGT AAATGATAG SEQ ID NO: 22 6.77.1-mod_ΔS Predicted Heavy Chain Protein Sequence    1 melglrwvfl vailegvqcE VQLVESGGGL VKPGGSLRLS CAASGFTFSS   51 YSMNWVRQAP GKGLEWVSSI SSSSSYIYYA DSVKGRFTIS RDNAKNSLYL  101 QMNSLRAEDT AVYYCARDGY SSGW-YYYYY GMDVWGQGTT VTVSSASTKG  151 PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA  201 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE  251 CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN  301 WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK  351 GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD  401 IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS  451 VMHEALHNHY TQKSLSLSPG K SEQ ID NO: 23 6.77.1-mod Kappa Light Chain Nucleotide Sequence    1 atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg   51 atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA  101 CTCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG  151 CTTAGTGATG GAAAGACCTA TTTGAATTGG TACCTGCAGA AGCCCGGCCA  201 GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC  251 CAGACAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC  301 AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAGTAT  351 ACAGCTTATG TGCAGTTTTG GCCAGGGGAC CAAGCTGGAG ATCAAACGAA  401 CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG  451 AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG  501 AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT  551 CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC  601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA  651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT  701 TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 24 6.77.1-mod Predicted Kappa Light Chain Amino Acid Sequence    1 mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL   51 LSDGKTYLNW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI  101 SRVEAEDVGV YSCMQSIQLM SSFGQGTKLE IKRTVAAPSV FIFPPSDEQL  151 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL  201 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO.25 7.16.6 Heavy Chain Nucleotide Sequence    1 atggactgga cctggagcat ccttttcttg gtggcagcag caacaggtgc   51 ccactccCAG GTTCAGCTGG TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG  101 GGGCCTCAGT GAAGGTCTCC TGCAAGGCTT CTGGTTACAC CTTTACCAGC  151 TATGGTATCA ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT  201 GGGATGGATC AGCGTTTACA GTGGTAACAC AAACTATGCA CAGAAGGTCC  251 AGGGCAGAGT CACCATGACC GCAGACACAT CCACGAGCAC AGCCTACATG  301 GACCTGAGGA GCCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG  351 AGAGGGTAGC AGCTCGTCCG GAGACTACTA TTACGGTATG GACGTCTGGG  401 GCCAAGGGAC CACGGTCACC GTCTCCTCAG CCTCCACCAA GGGCCCATCG  451 GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC  501 CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT  551 GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA  601 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG  651 CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA  701 ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA  751 CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC  801 AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG  851 TGGAGGTGCA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC  901 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA  951 GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001 ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101 ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251 TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401 GGGTAAATGA SEQ ID NO. 26 7.16.6 Predicted Heavy Chain Protein Sequence    1 mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS   51 YGINWVRQAP GQGLEWMGWI SVYSGNTNYA QKVQGRVTMT ADTSTSTAYM  101 DLRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS  151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL  201 QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP  251 PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY  301 VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL  351 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA  401 VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM  451 HEALHNHYTQ KSLSLSPGK SEG ID NO: 27 7.16.6_L Heavy Chain Nucleotide Sequence    1 atggactgga cctggagcat ccttttcttg gtggcagcag caacaggtgc   51 ccactccCAG GTTCAGCTGG TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG  101 GGGCCTCAGT GAAGGTCTCC TGCAAGGCTT CTGGTTACAC CTTTACCAGC  151 TATGGTATCA ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT  201 GGGATGGATC AGCGTTTACA GTGGTAACAC AAACTATGCA CAGAAGCTTC  251 AGGGCAGAGT CACCATGACC GCAGACACAT CCACGAGCAC AGCCTACATG  301 GACCTGAGGA GCCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG  351 AGAGGGTAGC AGCTCGTCCG GAGACTACTA TTACGGTATG GACGTCTGGG  401 GCCAAGGGAC CACGGTCACC GTCTCCTCAG CCTCCACCAA GGGCCCATCG  451 GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC  501 CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT  551 GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA  601 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG  651 CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA  701 ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA  751 CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC  801 AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG  851 TGGTGGTGGA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC  901 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA  951 GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001 ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101 ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251 TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1352 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401 GGGTAAATGA SEQ ID NO. 28 7.16.6_L Predicted Heavy Chain Protein Sequence    1 mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS   51 YGINWVRQAP GQGLEWMGWI SVYSGNTNYA QKLQGRVTMT ADTSTSTAYM  101 DLRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS  151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL  201 QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP  251 PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY  301 VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL  351 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA  401 VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM  452 HEALHNHYTQ KSLSLSPGK SEQ ID NO: 29 7.16.6_V Kappa Light Chain Nucleotide Sequence    1 atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg   51 atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA  101 CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG  151 CATACTGATG GAACGACCTA TTTGTATTGG TACCTGCAGA AGCCAGGCCA  201 GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC  251 CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC  301 AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAATAT  351 ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA  401 CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG  451 AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG  501 AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT  551 CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC  601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA  651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT  701 TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO: 30 7.16.6_V Predicted Kappa Light Chain Protein Sequence    1 mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL   51 HTDGTTYLYW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI  101 SRVEAEDVGV YYCMQNIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL  151 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL  201 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO: 31 7.16.6_S Kappa Light Chain Nucleotide Sequence    1 atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg   51 atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA  101 CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG  151 CATACTGATG GAACGACCTA TTTGTATTGG TACCTGCAGA AGCCAGGCCA  201 GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC  251 CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC  301 AGCCGGGTGG AGGCTGAGGA TGTTGGGATT TATTACTGCA TGCAAAGTAT  351 ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA  401 CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG  451 AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG  501 AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT  551 CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC  601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA  651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT  701 TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO: 32 7.16.6_S Predicted Kappa Light Chain Protein Sequence    1 mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL   51 HTDGTTYLYW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI  101 SRVEAEDVGI YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL  151 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL  201 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO: 33 7.16.6_VS Kappa Light Chain Nucleotide Sequence    1 atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg   51 atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA  101 CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG  151 CATACTGATG GAACGACCTA TTTGTATTGG TACCTGCAGA AGCCAGGCCA  201 GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC  251 CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC  301 AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAGTAT  351 ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA  401 CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG  451 AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG  501 AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT  551 CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC  601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA  651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT  701 TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO: 34 7.16.6_VS Predicted Kappa Light Chain Protein Sequence    1 mripaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL   51 HTDGTTYLYW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI  101 SRVEAEDVGV YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL  151 KSGTASVVCL LNNYYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL  201 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO: 35 9.8.2_ΔRGAYH, D Heavy Chain Nucleotide Sequence    1 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt   51 ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG  101 GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC  151 TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT  201 GGCAGTTATA TGGTATGATG GAAGTAATGA ATACTATGCA GACTCCGTGA  251 AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG  301 CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCG--  351 ---------- ---TTTGACT ACTGGGGCCA GGGAACCCTG GTCACCGTCT  401 CCTCAGCTTC CACCAAGGGC CCATCCGTCT TCCCCCTGGC GCCCTGCTCC  451 AGGAGCACCT CCGAGAGCAC AGCCGCCCTG GGCTGCCTGG TCAAGGACTA  501 CTTCCCCGAA CCGGTGACGG TGTCGTGGAA CTCAGGCGCC CTGACCAGCG  551 GCGTGCACAC CTTCCCGGCT GTCCTACAGT CCTCAGGACT CTACTCCCTC  601 AGCAGCGTGG TGACCGTGCC CTCCAGCAGC TTGGGCACGA AGACCTACAC  651 CTGCAACGTA GATCACAAGC CCAGCAACAC CAAGGTGGAC AAGAGAGTTG  701 AGTCCAAATA TGGTCCCCCA TGCCCATCAT GCCCAGCACC TGAGTTCCTG  751 GGGGGACCAT CAGTCTTCCT GTTCCCCCCA AAACCCAAGG ACACTCTCAT  801 GATCTCCCGG ACCCCTGAGG TCACGTGCGT GGTGGTGGAC GTGAGCCAGG  851 AAGACCCCGA GGTCCAGTTC AACTGGTACG TGGATGGCGT GGAGGTGCAT  901 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TTCAACAGCA CGTACCGTGT  951 GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAC GGCAAGGAGT 1001 ACAAGTGCAA GGTCTCCAAC AAAGGCCTCC CGTCCTCCAT CGAGAAAACC 1051 ATCTCCAAAG CCAAAGGGCA GCCCCGAGAG CCACAGGTGT ACACCCTGCC 1101 CCCATCCCAG GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG 1151 TCAAAGGCTT CTACCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 1201 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG 1251 CTCCTTCTTC CTCTACAGCA GGCTAACCGT GGACAAGAGC AGGTGGCAGG 1301 AGGGGAATGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC 1351 TACACACAGA AGAGCCTCTC CCTGTCTCTG GGTAAATGA SEQ ID NO: 36 9.8.2_ΔRGAYH, D Predicted Heavy Chain Protein Sequence    1 mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS   51 YGMHWVRQAP GKGLEWVAVI WYDGSNEYYA DSVKGRFTTS RDNSKNTLYL  101 QMNSLRAEDT AVYYCA---- -FDYWGQGTL VTVSSASTKG PSVFPLAPCS  151 RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL  201 SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPSCPAPEFL  251 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH  301 NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT  351 ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG  401 QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH  451 YTQKSLSLSL GK SEQ ID NO: 37 9.8.2-mod Kappa Light Chain Nucleotide Sequence    1 atggacatga gggtccctgc tcagctcctg gggctcctgc tgctctggct   51 ctcagtcgca ggtgccagat gtGACATCCA GATGACCCAG TCTCCATCCT  101 CCCTGTCTGC ATCTGTAGGA GACAGAGTCA CCATCACTTG CCAGGCGAGT  151 CAGGACATTA GCAACTATTT AAATTGGTAT CAGCAGAAAC CAGGGAAAGC  201 CCCTAAGCTC CTGATCTACG ATGCATCCAA TTTGGAAACA GGGGTCCCAT  251 CAAGGTTCAG TGGAAGTGGA TCTGGGACAG ATTTTACTTT CACCATCAGC  301 AGCCTGCAGC CTGAAGATAT TGCAACATAT TCCTGTCAAC ACTCTGATAA  351 TCTCATCACC TTCGGCCAGG GGACACGACT GGAGATTAAA CGAACTGTGG  401 CTGCACCATC TGTCTTCATC TTCCCGCCAT CTGATGAGCA GTTGAAATCT  451 GGAACTGCCT CTGTTGTGTG CCTGCTGAAT AACTTCTATC CCAGAGAGGC  501 CAAAGTACAG TGGAAGGTGG ATAACGCCCT CCAATCGGGT AACTCCCAGG  551 AGAGTGTCAC AGAGCAGGAC AGCAAGGACA GCACCTACAG CCTCAGCAGC  601 ACCCTGACGC TGAGCAAGGC AGACTACGAG AAACACAAAG TCTACGCCTG  651 CGAAGTCACC CATCAGGGCC TGAGCTCGCC CGTCACAAAG AGCTTCAACA  701 GGGGAGAGTG TTAGTGA SEQ ID NO: 38 9.8.2-mod Predicted Kappa Light Chain Protein Sequence    1 mdmrvpaqll gllllwlsva garcDIQMTQ SPSSLSASVG DRVTITCQAS   51 QDISNYLNWY QQKPGKAPKL LIYDASNLET GVPSRFSGSG SGTDFTFTIS  101 SLQPEDIATY SCQHSDNLIT FGQGTRLEIK RTVAAPSVFI FPPSDEQLKS  151 GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS  201 TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 

1-4. (canceled)
 5. A method for treating inflammatory disease in a patient comprising administering a therapeutically effective amount of a monoclonal antibody that specifically binds MAdCAM, or antigen-binding portion thereof, selected from the group consisting of: (a) an antibody comprising the variable region of the heavy chain of 6.22.2-mod_V (SEQ ID NO: 2) with or without signal sequence; (b) an antibody comprising the variable region of the light chain of 6.34.2-mod_SSQ, 6.34.2-mod_QT, or 6.34.2-mod_SSQ,QT (SEQ ID NOs: 8, 10, 12) with or without signal sequence; (c) an antibody comprising the variable region of the light chain of 6.67.1-mod_Y, 6.67.1-mod_TΔP, or 6.67.1-mod_Y,TΔP (SEQ ID NOs: 16, 18, 20) with or without signal sequence; (d) an antibody comprising the variable region of the heavy chain of 6.77.1-mod_AS (SEQ ID NO: 22) with or without signal sequence; (e) an antibody comprising the variable region of the heavy chain of 7.16.6 (SEQ ID NO: 26), or 7.16.6_L (SEQ ID NO: 28) with or without signal sequence; or (f) an antibody comprising the variable region of the light chain of 7.16.6_V, 7.16.6_S, or 7.16.6_VS (SEQ ID NOs: 30, 32, 34) with or without signal sequence; (g) an antibody comprising the variable region of the light chain of 7.16.6_V, 7.16.6_S, or 7.16.6_VS (SEQ ID NOs: 30, 32, 34) with or without signal sequences and the variable region of the heavy chain of 7.16.6 (SEQ ID NO: 26) or 7.16.6_L (SEQ ID NO: 28) with or without signal sequence; (h) an antibody comprising the variable region of the heavy chain of 9.8.2_ΔRGAYH,D (SEQ ID NO: 36) with or without signal sequence; and (i) an antibody comprising the variable region of the light chain of 9.8.2-mod (SEQ ID NO: 38) with or without signal sequence.
 6. The method of claim 5, wherein the inflammatory disease is inflammatory bowel disease, liver fibrosis, graft versus host disease, or uveitis.
 7. The method of claim 6, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.
 8. The method of claim 5, wherein the heavy chain C-terminal lysine of the antibody or portion thereof is cleaved.
 9. The method of claim 5, wherein the antibody or portion thereof comprises:(a) the variable region of the amino acid sequence set forth in SEQ ID NOs: 30, 32, or 34, without the signal sequence; and (b) the variable region of the amino acid sequence set forth in SEQ ID NO: 26 or 28, without the signal sequence.
 10. The method of claim 9, wherein the antibody comprises:(a) the light chain amino acid sequence set forth in SEQ ID NO: 30, 32, or 34, without the signal sequence; and (b) the heavy chain amino acid sequence set forth in SEQ ID NO: 26 or 28, without the signal sequence. 11-14. (canceled) 